α-Amylases (E.C. 3.2.1.1) hydrolyze internal α-1,4-glycosidic bonds of starch and starch-like polymers with the formation of dextrins and β-1,6-branched oligosaccharides. They are very much among the industrially most important enzymes. Thus, for example, α-amylases are employed in the production of glucose syrup, for the treatment of raw materials in the manufacture of textiles, for the production of adhesives or for the production of sugar-containing food or food ingredients. Another important field of use is the use as an active component in detergents and cleansers.
Since detergents and cleansers have mainly alkaline pH values, particular use is made here of α-amylases that are active in alkaline medium. These types of α-amylases are produced and secreted by microorganisms, i.e. fungi or bacteria, especially those of the genera Aspergillus and Bacillus. Starting from these natural enzymes, there is now quite a vast abundance of variants available which have been derived via mutagenesis and have specific advantages depending on the field of use.
Examples thereof are the α-amylases of Bacillus licheniformis, B. amyloliquefaciens and B. stearothermophilus and their improved developments for the use in detergents and cleansers. The B. licheniformis enzyme is available from Novozymes under the name Termamyl® and from Genencor under the name Purastar® ST. Products of further development of this α-amylase can be obtained from Novozymes under the trade names Duramyl® and Termamyl® ultra, from Genencor under the name Purastar® OxAm and from Daiwa Seiko Inc., Tokyo, Japan, as Keistase®. The B. amyloliquefaciens α-amylase is sold by Novozymes under the name BAN®, and variants derived from the B. stearothermophilus α-amylase are sold under the names BSG® and Novamyl®, likewise by Novozymes.
Examples of α-amylases from other organisms are further developments of the Aspergillus niger and A. oryzae α-amylases, obtainable under the trade name Fungamyl® from Novozymes. Another commercial product is Amylase-LT®, for example.
Mention may further be made of the Bacillus sp. A 7-7 (DSM 12368) α-amylase disclosed in the application WO 02/10356 A2 and of the B. agaradherens (DSM 9948) cyclodextrin glucanotransferase (CGTase) described in the application WO 02/44350 A2. In addition, for example, the applications WO 03/002711 A2 and WO 03/054177 A2 define sequence spaces of α-amylases, all of which could be suitable in principle for corresponding applications.
The application DE 10309803.8, which has not been prepublished, for example, describes point mutations for improving the activity of said enzymes in alkaline medium. According to this application, amino acid substitutions suitable here are those in positions 13, 32, 194, 203, 230, 297, 356, 406, 414 and/or 474, according to the numbering of the unprocessed B. amyloliquefaciens α-amylase.—These positions are according to the numbering of the unprocessed Bacillus sp. A 7-7 (DSM 12368) α-amylase (WO 02/10356 A2) L13, T36, W198, S201, I208, A235, D302, D361, H408, K416 and N476, respectively, with the following, particularly effective substitutions: 13P, 32A, 194R, 197P, 203L, 230V, 297D, 356D, 406R, 414S and 474Q.
Another example of point mutagenesis on α-amylases is the application WO 00/22103 A1 which discloses polypeptides, inter alia also α-amylase variants, containing mutagenized surface amino acids. The purpose of this mutagenesis was to reduce the immunogenicity and/or allergenicity caused by these molecules.
Fusion products of α-amylases for the use in detergents and cleansers have also been described. Thus, for example, the application WO 96/23874 A1 discloses hybrids of the α-amylases of Bacillus licheniformis, B. amyloliquefaciens and B. stearothermophilus. According to the teaching of this application, such hybrid amylases may be prepared for determining the three-dimensional structure of said amylases, in order to use said structure for detecting important positions for enzymic activity. Further developments in this respect are the applications WO 97/41213 A1 and WO 00/60059 A2, which report numerous α-amylase variants whose respective performances have been improved. The application WO 03/014358 A2 discloses special hybrid amylases of B. licheniformis and B. amyloliquefaciens. 
The three applications WO 96/23873 A1, WO 00/60060 A2 and WO 01/66712 A2, which are the basis of the commercial product Stainzyme® from Novozymes, constitute another important prior art. All the variants obtainable by point mutagenesis which are specified in each of these applications have altered enzymic properties and are therefore claimed or described for the use in detergents and cleansers. WO 96/23873 A1 makes mention of, in some cases two or more, point mutations in more than 30 different positions in four different wild type amylases. They apparently have altered enzymic properties with regard to thermal stability, oxidation stability and calcium dependence. They include point mutations in the following positions, each of which is stated with respect to the Bacillus sp. NCIB 12512 α-amylase: substitution of oxidizable amino acids in positions M9, M10, M105, M202, M208, M261, M309, M382, M430 or M440, preferably M91L; M10L; M105L; M202L,T,F,I,V; M208L; M261L; M309L; M382L; M430L and M440L; deletions of F180, R181, G182, T183, G184 and/or K185; and additionally the substitutions K269R; P260E; R124P; M105F,I,L,V; M208F,W,Y; L217I; V206I,L,F; Y243F; K108R; K179R; K239R; K242R; K269R; D163N; D188N; D192N; D199N; D205N; D207N; D209N; E190Q; E194Q or N106D.
The application WO 00/60060 A2 likewise specifies a multiplicity of possible amino acid substitutions in 10 different positions, on the basis of two very similar α-amylases from two different microorganisms, with the same numbering α-amylases AA349 and AA4560). These are the following sequence variations: R181*, G182*, D183*, G184*; N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 1206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; and/or R181A,N,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V. This development too is against the background of improving performance via mutations.
WO 01/66712 A2, finally, refers to 31 different amino acid positions which are partially identical to the ones mentioned above and which have been mutated in either of the two α-amylases specified in the application WO 00/60060 A2 and which are said to improve aspects of both performance and stability. These are point mutations in the following positions: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 and N484, again as defined via α-amylase AA560, i.e. also according to the numbering of the letter. Among these, the following variants are said to be particularly advantageous: Delta G184; Delta (R181-G182); Delta (D183-G184); R28N,K; S94K; R118K; N125A,R,K; N174D; R181Q,E,K; G186R; W189R,K; N195F; M202L,T; Y298H,F; N299A; K302R, S303Q, N306G,D,R,K; R310A,K,Q,E,H,D,N; N314D; R320K; H324K; E345R,D,K,N; Y396F; R400T,K; W439R; R444K; N445K,Q; K446N; Q449E; R458K; N471E and N484Q.
This latter application further describes polypeptide crystals, in particular those of enzymes, that might be improved with respect to their resolution capacity in that, in the molecules located next to the actual molecules of interest in the crystal in question and interacting therewith, those amino acids which are located within a distance of 6.0 Å to the polypeptide of interest could be mutated. This would apply in particular to Termamyl-like enzymes located next to other or identical Termamyl-like enzymes in a crystal; here in particular to a distance of less than 3.5 Å. This would concern positions 19, 20, 21, 22, 25, 28, 29, 53, 76, 84, 87, 90, 93, 94, 124, 125, 126, 128, 142, 144, 156, 157, 158, 159, 160, 161, 170, 171, 172, 173, 174, 175, 183, 184, 185, 186, 187, 188, 189, 190, 193, 195, 196, 197, 209, 212, 226, 229, 256, 257, 258, 259, 280, 281, 298, 299, 300, 302, 303, 304, 305, 306, 310, 311, 314, 319, 320, 321, 322, 341, 345, 405, 406, 408, 444, 447, 448, 449, 463, 464, 465, 466 and 467; and positions 22, 25, 28, 76, 94, 125, 128, 158, 160, 171, 173, 174, 184, 189, 209, 226, 229, 298, 299, 302, 306, 310, 314, 320, 345, 405, 447 and 466 for the shorter distance.
All of these applications regarding point mutagenesis share the fact that the point of stability is also evaluated under the aspect of a good performance in the corresponding field of use. This is because the stability maintained during storage and usage of α-amylases, for example within the context of detergent formulations, results in a high performance or in a performance as constantly high as possible with corresponding usage. An increase in stability, in particular to aggregate formation, above all in the course of workup, is not described.
The purpose of increasing stability is pursued by numerous applications describing special enzyme stabilizers. These additional ingredients cause a protein and/or enzyme present in corresponding agents to be protected from damage such as, for example, inactivation, denaturation or decay, particularly during storage. Thus, reversible protease inhibitors form a group of stabilizers. Others, for example polyols, stabilize to physical influences such as freezing, for example. Other polymeric compounds such as acrylic polymers and/or polyamides stabilize the enzyme preparation inter alia to pH fluctuations. Reducing agents and antioxidants increase stability of the enzymes to oxidative decay.
Compounds of these kind are added to the enzymes both during application and in the course of their work-up, which is particularly important, if a previously present stabilizer has been removed together with the other contaminations in a component step of said workup, for example a precipitation.
The prior art regarding the improvement in stability of α-amylases can be summarized as follows: a multiplicity of α-amylase variants have been developed via point mutagenesis, with the aim of these developments having mainly been that of improving the performance of said α-amylase variants. This category also includes those variants which have been stabilized with regard to denaturing agents such as bleaches or surfactants, since in these cases, the desired performance of the enzyme is optimized. In other cases, additional compounds which are overall referred to as stabilizers are mainly used for increasing stability or maintaining the physicochemical conformation.
A previously less regarded aspect in enzyme development is that of stabilizing the molecules per se in such a way that they have increased stability over the wild type molecule even during their workup. An additional advantageous effect thereof would be that this increased stability should also benefit the intended later usage of the enzyme in question.
The necessity for this is particularly evident in the case of α-amylases. At least some of these tend, especially during production and workup, to form multimers, specifically in the form of amorphous aggregates, which precipitate irreversibly. As a result, the activities in question are lost even during workup. The work-up process includes all steps of industrial production, starting from isolating the enzyme in question, in particular the fermentation media common in biotechnological production, via the following washing and separation steps (for example by precipitation) and concentration up to formulation, for example granulation. In particular, those substeps in which the enzyme is present in solutions with comparatively high concentrations are critical here, because, seen statistically, more frequent contacts occur here between the molecules than at lower concentrations. However, the aggregate formation may also occur during storage of α-amylase-containing agents or during application, for example when used as active ingredient in washing or cleaning processes.
This problem can go as far as individual α-amylases, although they can be produced and studied on a laboratory scale, refusing to be produced on an industrial scale with the aid of generally common methods. This is then also referred to as said enzymes having low process stability, meaning a large variety of possible processings and uses. For example, Bacillus sp. A 7-7 (DSM 12368) α-amylase exhibits a greater tendency toward multimerization than the native B. licheniformis α-amylase. Approaches to eliminate this type of instability, that is to say to reduce the tendency toward multimerization, would only enable such enzymes in the first place to be accessible to production on an industrial scale and thus to the large variety of fields of use in industrially relevant quantities.